Arrays of surface-bound binding agents may be used to detect the presence of particular targets, e.g., biopolymers, in solution. The surface-bound probes may be oligonucleotides, peptides, polypeptides, proteins, antibodies or other molecules capable of binding with target molecules in solution. Such binding interactions are the basis for many of the methods and devices used in a variety of different fields, e.g., genomics (in sequencing by hybridization, SNP detection, differential gene expression analysis, identification of novel genes, gene mapping, finger printing, etc.) and proteomics.
One typical array assay method involves biopolymeric probes immobilized in an array on a substrate, such as a glass substrate or the like. A solution containing analytes that bind with the attached probes is placed in contact with the array substrate, covered with another substrate such as a coverslip or the like to form an assay area and placed in an environmentally controlled chamber such as an incubator or the like. Usually, the targets in the solution bind to the complementary probes on the substrate to form a binding complex. The pattern of binding by target molecules to biopolymer probe features or spots on the substrate produces a pattern on the surface of the substrate and provides desired information about the sample. In certain instances, the target molecules are labeled with a detectable tag such as a fluorescent tag or chemiluminescent tag. The resultant binding interaction or complexes of binding pairs are then detected and read or interrogated, for example by optical means, although other methods may also be used. For example, laser light may be used to excite fluorescent tags, generating a signal only in those spots on the biochip that have a target molecule and thus a fluorescent tag bound to a probe molecule. This pattern may then be digitally scanned for computer analysis.
As such, optical scanners play an important role in many array based applications. Optical scanners act like a large field fluorescence microscope in which the fluorescent pattern caused by binding of labeled molecules on the array surface is scanned. In this way, a laser induced fluorescence scanner provides for analyzing large numbers of different target molecules of interest, e.g., genes/mutations/alleles, in a biological sample.
For each pixel of a scan, a light detector (e.g., a photomultiplier tube) typically detects light emitted from the surface of a microarray, and outputs an analog signal that changes in amplitude according to the amount of emitted light entering the detector. This analog signal is usually sampled and digitized using an analog-to-digital converter (A/D converter) and integrated using a digital signal processor (DSP) to provide data, e.g., a numerical evaluation of the brightness of the pixel. This data is usually stored and analyzed at a later date.
However, current data processing methodologies are limited in their capacity to obtain reliable data from every pixel of a scan because there is a limitation to the amplitude of input signals of system components in current signal detection systems. For example, the signal output of a detector may exceed the signal input range of a current-to-voltage converter, or the output of a current-to-voltage converter may exceed the signal input range for an analog-to-digital converter. Accordingly, for many bright areas of a scan, the analog signal produced by a light detection system may be partially or fully “saturated”, i.e., at the maximum amplitude. Because of this limitation, an integrated signal representing a pixel may not always accurately represent the amount of light entering the detector. Despite this limitation, saturated signals are typically digitized and integrated using similar methods to those for non-saturated signals, leading to inaccurate data.
Accordingly, there is a great need for a signal integration system that can increase the accuracy of data obtained from a saturated pixels. The present invention meets this, and other, needs.
Literature of interest includes: published U.S. patent applications: 20030168579, 20030165871, 20040064264, 20040023224, 20040021911, 20030203371 and 20030168579; and Cheung et al., Nature Genetics 1999, 21: 15-19.